1. Field of the Invention
The present invention relates to a method of SNP (Single-nucleotide polymorphism) detection by using gene detection technique. More particularly, the present invention relates to a method of SNP detection by using gene detection technique in bead-based microfluidics with a well-controlled temperature-gradient region inside the microchannels.
2. Description of Related Art
Single nucleotide polymorphisms (SNPs) are one of the most common types in genetic variations, estimated to occur at 1 out of every 1,000 bases in the human genome, which means more than 10 million points of SNPs occurring across the human genome. SNPs are important markers that link sequence variations to phenotypic changes; such researches are expected to advance the understanding of human physiology and to elucidate the molecular bases of diseases. To date, a great deal of work has been devoted to developing accurate, rapid, and cost-effective technologies for SNP genotyping. The genotyping procedures typically involve the amplification of allele-specific products for SNP of interest, followed by the genotype detection techniques, such as enzymatic ligation, enzymatic cleavage, primer extension, split DNA enzymes G-quadruplex, sequencing, pyrosequencing, and mass spectroscopy. All of these techniques utilize enzymes, molecular beacon, or fluorescent dyes to label the DNA probes, leading to the requirement of high reagent cost or complicate procedures.
On the other hand, the dynamic allele-specific hybridization (DASH) technique has drawn great attention in SNP genotyping since it doesn't require the complex and expensive modification procedures on enzymes or fluorescent molecules. A conventional DASH procedure is described as follows. A target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated strand is rinsed away with alkali. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This forms a duplex DNA region that interacts with a double strand-specific intercalating dye. Upon excitation, the dye emits fluorescence proportional to the amount of double stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing (or “melting”) temperature of the probe-target duplex. When performed under appropriate buffer and dye conditions, a single-base mismatch between the probe and the target results in a dramatic lowering of melting temperature (Tm) that can be easily detected.
Furthermore, miniaturized devices, for instance microfluidic or lab-on-a-chip devices, have brought many advantages over their analogues at the macroscale, including portability, reduced sample consumption, rapid reaction times, and high throughput. The microbeads can serve as a vehicle to immobilize the target biomolecules, carry the biomolecules for a series of reactions. Bead-based microfluidic devices thus can significantly simplified the tedious and labor-intensive washing procedures of traditional DNA/RNA purification and double-stranded DNA isolation process.
In recent years, the development in microfluidics further promotes melting analysis within the aforesaid miniature device, which not only can reduce the reagent cost but also provide the possibility point-of-care molecular diagnosis. The DNA melting analysis in microfluidic device usually resorts to solid or liquid phase on sample preparations, in which either DNA immobilization on channel surfaces is required or only single analysis is allowed. However, some related arts, such as Russom et al, “rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms” (Anal. Chem. 78, 2006 3515867), provide a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis was based on dynamic allele-specific hybridization (DASH). The DNA duplexes were conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors, and a melting curve was obtained. However, those related arts failed to teach the temperature gradient region enhancing the measurement sensitivity and accuracy. To address these limitations, a bead-based melting analysis with temperature gradient configuration is required.